CircRANBP17 modulated KDM1A to regulate neuroblastoma progression by sponging miR-27b-3p

Abstract Neuroblastoma (NB) is a common childhood cancer. Circular RNA RAN binding protein 17 (circRANBP17) has been identified to participate in diverse tumor progression. This study aims to explore the function and mechanism of circRANBP17 in NB. The levels of circRANBP17, miR-27b-3p and KDM1A in NB tissues and cells were measured by qRT-PCR. Mouse model assay was performed to investigate the effect of circRANBP17 knockdown on tumor formation in vivo. The levels of circRANBP17 and KDM1A were significantly up-regulated, and the level of miR-27b-3p was strikingly down-regulated in NB tissues and cells (SK-N-SH and SK-N-AS). Functional studies indicated that miR-27b-3p inhibitor mitigated the inhibitory effects on cell proliferation, migration, invasion and the promoting effect on cell apoptosis in SK-N-SH and SK-N-AS cells induced by circRANBP17 knockdown. Also, miR-27b-3p regulated NB cell malignancy by targeting KDM1A. Further studies revealed that miR-27b-3p inhibitor reversed the low expression of KDM1A induced by circRANBP17 knockdown. In support, circRANBP17 knockdown led to inhibition of tumor formation in vivo. In conclusion, circRANBP17 modulated KDM1A to promote cell proliferation, migration, invasion and restrain cell apoptosis in NB by sponging miR-27b-3p, and the new regulatory network may provide a theoretical basis for the further study of NB.


Introduction
Neuroblastoma (NB) originates from the developing sympathetic nervous system, characterized by heterogeneous clinical behavior [1,2]. Despite the improvement of treatment strategies in recent years, the survival rate of highrisk NB remains 40-50% [3,4]. The main obstacle to NB treatment is high relapse and mortality [5]. Thus, it is necessary to further understand the mechanism behind NB progression.
Circular RNA (circRNA) is a conserved non-coding RNA and can function as an oncogene or tumor-repressing factor to regulate tumor progression [6]. Dysregulation of circRNAs is associated with cancers including NB. Zhang et al. revealed that circRNA cut like homeobox 1 (circCUX1) was strikingly up-regulated in NB tissues as well as cells, and its knockdown repressed tumor growth in vivo [7]. Fang and his colleagues reported that there were 29 dysregulated circRNAs in NB classes [8]. In another study, the malignant behaviors of NB cells were found to be related to circ_0132817/miR-432-5p/nucleolar protein 4 like (NOL4L) axis [9]. circRNA RAN binding protein 17 (circRANBP17) was an oncogene in nasopharyngeal carcinoma [10]. Moreover, we found that circRANBP17 was one of the top eight up-regulated circRNAs involved in the alteration in response to neuronal depolarization [11]. However, the mechanism of circRANBP17 was still unclear in NB.
MiRNAs, a class of non-coding RNAs, have been identified as tumor suppressors or carcinogens in many tumors including NB [12]. Zhu et al. indicated that miR-186-5p was down-regulated in NB patients, and its overexpression induced cell apoptosis and impeded cell proliferation; miR-186-5p overexpression inhibited tumor growth in vivo [13]. Another investigation about NB confirmed that miR-144-3p, weakly expressed in NB tissues, inhibited NB tumor progression in vitro [14]. The lysinespecific histone demethylase 1A (KDM1A, also named LSD1 or AOF2), a histone demethylase, is involved in many cancers [15,16]. Previous evidence confirmed that KDM1A promoted cell proliferation as well as motility in NB [17]. As a result, the biological mechanisms involving the function of miR-27b-3p and KDM1A in NB remain poorly understood.
Herein, we hypothesized that circRANBP17 induced KDM1A expression by interacting with miR-27b-3p to regulate NB cell phenotypes. We analyzed circRANBP17 expression in NB to determine its role in NB progression. The evidence from our study unraveled that circRANBP17 regulated KDM1A by targeting miR-27b-3p, so as to stimulate NB processes through regulation of cell proliferation, metastasis and apoptosis, shedding light on the mechanism of NB.

Tissue sample
With the approval of the Ethics Committee of The Second Affiliated Hospital of Soochow University, 35 pairs of NB tissues and corresponding adjacent normal tissues were collected from The Second Affiliated Hospital of Soochow University. All tissues were frozen at −80°C. Informed consent was provided by NB patients or their guardians.

Quantitative real-time PCR (qRT-PCR)
One-step extraction method was utilized for isolation of total RNA with Trizol (Thermo Fisher Scientific). The specific steps were performed following the standard instructions. The concentration of RNA sample was detected using UV-3100PC spectrophotometer. Reverse transcription was conducted using cDNA synthesis kit (TaKaRa, Dalian, China). For the quantification of gene expression, SYBR Premix Ex Taq II (TaKaRa) was mixed with cDNA and reacted on qRT-PCR machine (Bio-Rad, Hercules, CA, USA) according to the standard methods. The relative expressions of circRANBP17, miR-27b-3p and KDM1A were calculated by the 2 −ΔΔCt method.

Nucleocytoplasmic separation
NB cells were trypsinized and centrifuged at low speed. After washing with phosphate buffer solution, the cell pellet was placed on ice. Then, nucleocytoplasmic separation was performed with a PARIS™ Kit (Thermo Fisher Scientific) as instructed. The RNA from cytoplasm and nucleus was isolated and qRT-PCR was applied to quantify circRANBP17 expression. β-actin and U6 served as controls.

Identification of circRNA stability
The NB cells grown in 12-well plates were subjected to incubation with Actinomycin D (Amresco, Solon, OH, USA) for 0, 4, 8, 12, and 24 h. These cells were subsequently lysed and circRANBP17 consent was quantified by qRT-PCR. Linear RANBP17 acted as a reference.
For 5-ethynyl-29-deoxyuridine (EdU) assay, SK-N-SH and IMR-32 cells were seeded in 12-well plates (1 × 10 5 cells per well) and treated with test compounds. Meanwhile, 96-well plates were incubated with EdU-labeled medium for 2 h. After that, the cells were seeded into the 96-well plates, and the EdU-positive cells were analyzed by using cell proliferation detection kit (Ribobio, Guangzhou, China) following the guidebook.
For cell colony formation assay, the SK-N-SH and IMR-32 cells were grown in 6-well plates (500 cells per well). In brief, plasmids or oligonucleotides were transfected into the cells according to different aims. About 2week culture later, the cells were incubated with paraformaldehyde (Phygene, Fuzhou, China) and crystal violet (Phygene), respectively. At last, the number of colonies greater than 1 mm in diameter was calculated to assess cell proliferation.

Wound-healing assay
NB cells were cultured in culture plates after various treatments. The cell wounds were created when cells were filled with the lower surface of the plates. Then, floated cells and debris were discarded, and the cells were cultured for 24 h. At last, the distance migrated by the cells was determined by analyzing the width of wounds under low-power microscope (Nikon, Tokyo, Japan).

Transwell assay
Transwell chambers were used to analyze cell migratory ability as per the method descripted by the manufacturer (Corning Tewksbury, MA, USA). In brief, NB cells were added into the upper chamber with serum-free DMEM. As chemoattractants, DMEM containing 10% FBS was placed into the lower chambers. After incubated for 24 h, the cells were fixed with 4% methanol, and then stained with 0.1% crystal violet. Cells were counted under a microplate (Nikon). The invasion assay was implemented based on the above methods except the cells were cultured in Matrigel chambers.

Cell apoptosis assay
After incubated for 24 h, the NB cells were digested with 0.25% trypsin. Then the fluorescence intensity of cells stained with Annexin V-FITC and propidium iodide were detected using Apoptosis Detection Kit (Solarbio, Beijing, China) to measure the apoptosis rate on a flow cytometry (Thermo Fisher Scientific).

RNA immunoprecipitation (RIP) assay
RIP assay was conducted using RIP assay Kit (Millipore, Bradford, MA, USA) according to the manual. In brief, the cell lysed fragment samples were incubated with magnetic beads conjugated with anti-Ago2 (Millipore) or anti-IgG (Millipore) antibody. Following that, the level of circRANBP17 was detected by qRT-PCR.

RNA pull-down assay
Biotinylated wild-type and mutant circRANBP17 (Bio-circRANBP17 WT and Bio-circRANBP17 MUT), and negative control (Bio-NC) were transfected into NB cells, respectively. After 48 h incubation, the cells were lysed using RIP lysis buffer, and incubated with streptavidincoupled beads (Invitrogen) for 4 h. At last, qRT-PCR was used to determine miR-27b-3p expression.

Mouse model assay
The tumor xenograft investigation was performed with the approval of the Ethics Committee of The Second Affiliated Hospital of Soochow University. BALB/c nude mice (female; 5-6 weeks of age) were purchased from Charles River (Beijing, China), and arbitrarily divided into sh-NC and sh-circRANBP17 groups (N = 5 per group). Exclusion criteria were based on the differences of tumor volume and weight. The 5 × 10 6 SK-N-SH cells stably expressing the small hairpin RNAs against circRANBP17 or negative control were adjusted to suitable concentration, and then subcutaneously injected into the right side of mice back. The volume of the forming tumors was measured every 1 week since the seventh day after injection as per volume (mm 3 ) = width 2 × length × 1/2. After 35 days, these mice were euthanatized, and the forming tumors were harvested for further analysis.

Statistical analysis
All data were analyzed using GraphPad Prism 7. Quantitative analysis was repeated for three times, and presented as mean ± standard deviation. Difference analysis in two groups was examined by Student's t-test, and in more than three groups by one-way analysis of variance. Statistical significance is considered when P < 0.05.
Ethics approval and consent to participate: Written informed consents were obtained from all participants and this study was permitted by the Ethics Committee of The Second Affiliated Hospital of Soochow University.

CircRANBP17 is significantly up-regulated in NB tissues and cells
To explore the role of circRANBP17 in NB, the relative expression of circRANBP17 was first measured by qRT-PCR. The results showed that the level of circRANBP17 was apparently increased in NB tissues in contrast to its expression in normal brain tissues (Figure 1a). Also, cir-cRANBP17 was strikingly elevated in BE(2)-C, SK-N-AS, SK-N-SH and IMR-32 cells compared to that in HUVEC cells (Figure 1b). SK-N-SH and IMR-32 cells were employed for subsequent study as the higher expression of circRANBP17 in them. Additionally, we found that circRANBP17 was mainly located in cytoplasm of NB cells (Figure 1c and d). Furthermore, the data showed the high stability of circ-RANBP17 (Figure 1e and f). These results indicated that circRANBP17 was remarkably augmented in NB tissues and cells.

CircRANBP17 knockdown restrains cell proliferation, migration, invasion and induces cell apoptosis in SK-N-SH and IMR-32 cells
To illustrate the function of circRANBP17 in NB, circRANBP17 was silenced for the further exploration. The knockdown efficiency was confirmed by qRT-PCR, indicated by the evident down-regulation of circRANBP17 in SK-N-SH and IMR-32 cells (Figure 2a and b). Furthermore, the results exhibited that the cell proliferative capacity was inhibited after circRANBP17 knockdown (Figures 2c-e and 3a). Additionally, migration ability and invasion ability were all decreased in circRANBP17-knockdowned SK-N-SH and IMR-32 cells related to that in si-NC group (Figure 3b-d).
Flow cytometry presented that the apoptosis rate was dramatically enhanced in SK-N-SH and IMR-32 cells transfected with si-circRANBP17 in comparison with that in si-NC group (Figure 3e). Since Bcl-2 acted as inhibitor and Bax, Cleaved caspase 3 functioned as proapoptotic factors in cell apoptosis [19], we investigated the effect of circRANBP17 silencing on the protein levels of Bcl-2, Bax and Cleaved caspase 3 by western blot assay. The results showed that the protein level of Bcl-2 was apparently decreased and the protein levels of Bax as well as Cleaved caspase 3 were notably increased in SK-N-SH and IMR-32 cells (Figure 3f). Taken together, the depletion of circRANBP17 inhibited cell proliferation, migration, invasion and promoted cell apoptosis in SK-N-SH and IMR-32 cells.

miR-27b-3p negatively interacts with circRANBP17
To investigate the potential mechanism of circRANBP17 in NB, starbase tools online database was used to search   the putative target of circRANBP17. The miRNAs with circRANBP17-binding sites and related to NB progression were analyzed by qRT-PCR after circRANBP17 depletion in SK-N-SH and IMR-32 cells. As shown in Figure A1a and b, circRANBP17 depletion significantly up-regulated miR-195-5p, miR-432-5p and miR-27b-3p, especially up-regulated miR-27b-3p. Thus, miR-27b-3p was chosen for the present study. The results showed that miR-27b-3p had complementary sequence of circRANBP17 (Figure 4a). Dual-luciferase reporter assay showed that the transfection of miR-27b-3p mimics resulted in the distinct reduction of luciferase activity of circRANBP17 WT, while luciferase activity had no apparent change in miR-NC group; however, the luciferase activity of circRANBP17 MUT reporter had no significant fluctuation in SK-N-SH and IMR-32 cells transfected with miR-27b-3p or miR-NC (Figure 4b and c). Moreover, the RIP assay indicated that circRANBP17 was notably enriched by Ago2 antibody in comparison with that in IgG control group (Figure 4d). RNA pull-down assay displayed that miR-27-3p was enriched in Bio-circRANBP17 WT group as compared with its expression in Bio-circRANBP17 MUT group (Figure 4e). In addition, the level of miR-27b-3p was evidently decreased in NB tissues and cells compared to that in their matched controls (Figure 4f and g). After analyzing the relationship between miR-27b-3p expression and clinicopathologic features of NB patients, we found that miR-27b-3p expression was associated with tumor-node-metastasis grade ( Table A1). The scatter diagram showed that the level of circRANBP17 was negatively correlated with miR-27b-3p (Figure 4h). Further data displayed that miR-27b-3p was obviously increased in SK-N-SH and IMR-32 cells with the transfection of si-circRANBP17, which was attenuated after miR-27b-3p depletion (Figure 4i). These data manifested that miR-27b-3p was a direct target of circRANBP17.

miR-27b-3p inhibitor restrains the inhibitory effects on cell proliferation, migration, invasion and the promotion effect on cell apoptosis in SK-N-SH and IMR-32 cells induced by circRANBP17 knockdown
To confirm the function of circRANBP17 and miR-27p-3p in NB, si-circRANBP17 and in-miR-27b-3p were co-transfected into SK-N-SH and IMR-32 cells. The data presented that the cell proliferative capacity was inhibited in circRANBP17-knockdown SK-N-SH and IMR-32 cells, which was rescued after miR-27b-3p absence (Figure 5a-d).
Additionally, migration and invasion ability were all obviously down-regulated in si-circRANBP17-transfected SK-N-SH and IMR-32 cells, while the emergence of in-miR-27b-3p reversed the inhibitory effect (Figure 5e-g). However, the promoting effect of circRANBP17 absence on the apoptosis of SK-N-SH and IMR-32 cells was remitted by reduced expression of miR-27b-3p (Figure 5h-j). For instance, cir-cRANBP17 absence led to the increase of apoptotic rate and the protein expression of Bax as well as Cleaved caspase 3, and the inhibition of Bcl-2 expression; however, these effects were relieved after miR-27b-3p depletion. These results revealed that miR-27b-3p inhibitor attenuated the inhibitory effects on cell proliferation, migration, invasion and the promotion effect on cell apoptosis in SK-N-SH and IMR-32 cells caused by cir-cRANBP17 knockdown.

KDM1A negatively interacts with miR-27b-3p
To explore the underlying mechanism of miR-27b-3p in NB, DIANA tools online database was used to search the putative target of miR-27b-3p. The results exhibited that KDM1A 3′-UTR had complementary binding sites of miR-27b-3p (Figure 6a). The luciferase activity of KDM1A 3′-UTR-WT reporter was dramatically down-regulated in SK-N-SH and IMR-32 cells transfected with miR-27b-3p mimics, while the luciferase activity of KDM1A 3′-UTR-MUT reporter had no significant change (Figure 6b and c). In addition, the level of KDM1A was elevated in NB tissues and cells (Figure 6d and f). The scatter diagram indicated that KDM1A was negatively correlated with miR-27b-3p (Figure 6e). The protein level of KDM1A was drastically down-regulated in SK-N-SH and IMR-32 cells transfected with miR-27b-3p mimics, whereas the effect was restored after KDM1A overexpression (Figure 6g). Based on the above results, the relationship among cir-cRANBP17, miR-27b-3p and KDM1A was further explored. The qRT-PCR and western blot assay results exhibited that KDM1A was effectively down-regulated in circRANBP17knockdowned SK-N-SH and IMR-32 cells, while the level of KDM1A was reverted after transfection with in-miR-27b-3p ( Figure 6h). These data unraveled that miR-27b-3p inhibitor reversed the low expression of KDM1A induced by cir-cRANBP17 knockdown. (d) Enrichment of circRANBP17 by Ago2 or IgG antibody was performed by RIP assay. (e) RNA pull-down assay was carried out to confirm the association of miR-27b-3p and circRANBP17. The level of miR-27b-3p in NB tissues (f) and cells (g) was measured by qRT-PCR.

KDM1A overexpression mitigated the inhibitory effects on cell proliferation, migration, invasion and the promotion effect on cell apoptosis in SK-N-SH and IMR-32 cells caused by miR-27b-3p
To further explore the biological effect of miR-27b-3p and KDM1A in NB, miR-27b-3p and KDM1A were co-transfected into SK-N-SH and IMR-32 cells. Our data showed that cell proliferative capacity, migration ability and invasion ability were significantly decreased in SK-N-SH and IMR-32 cells, while the inhibitory effects were reversed by the emergence of KDM1A (Figure 7a-g). However, the cell apoptosis rate exhibited the opposite trend (Figure 7h). The western blot assay indicated that the protein level of Bcl-2 was strikingly decreased in SK-N-SH and IMR-32 cells transfected with miR-27b-3p mimics, while the level was regained in SK-N-SH and IMR-32 cells co-transfected with miR-27b-3p mimics and KDM1A; the protein levels of Bax, Cleaved caspase 3 showed the opposite trend (Figure 7i and j). These results implied that KDM1A overexpression alleviated the inhibitory effects on cell proliferation, migration, invasion and the promoting effect on cell apoptosis in SK-N-SH and IMR-32 cells induced by miR-27b-3p.

CircRANBP17 knockdown inhibited NB cell malignancy in vivo
The in vivo assay was further performed to demonstrate the effect of circRANBP17 depletion on tumor formation. The data showed that reduced expression of circRANBP17 led to the delayed tumorigenesis (Figure 8a and b). Then, the study found that circRANBP17 and KDM1A expressions were down-regulated, while miR-27b-3p expression was up-regulated in the primary tumors from cir-cRANBP17-knockdowned SK-N-SH cells (Figure 8c-e) as compared with controls. Also, the positive expression rate of both KDM1A and Ki-67 was lower in sh-circRANBP17 group than in sh-NC group (Figure 8f). These data demonstrated that circRANBP17 depletion inhibited NB cell malignancy in vivo.

Discussion
NB is a common malignant extracranial cancer in children. As reported, circRNAs play key roles in different types of tumors including NB. Based on the absent data about circRANBP17 in NB malignant progression, the study was organized to reveal circRANBP17's role in NB and its underlying mechanism. As a result, we found that circRANBP17 could regulate NB progression via miR-27b-3p/KDM1A axis. Accumulating evidence reported that NB malignant progression involved the altered expression of circRNAs, such as circ_0133622 [20], circRNA generated from AGO2 gene (circAGO2) [21] and circRNA TBC1 domain family member 4 (circTBC1D4) [22], which suggested the importance of circRNAs in NB progression. CircRANBP17, a novel circRNA, has been found to function as an oncogene in nasopharyngeal carcinoma [10]. In this work, we found circRANBP17 was located in cytoplasm of NB cells, and augmented in the clinical NB specimens and cells. Importantly, circRANBP17 silencing inhibited cell proliferative and migratory behaviors, and increased cell apoptotic rate. Besides, the altered levels of apoptosis-related Bcl-2, Bax and Cleaved caspase 3 also validated that the depletion of circRANBP17 promoted cell apoptosis in NB cells. Furthermore, in vivo assay also confirmed the repressing effect of circRANBP17 knockdown on tumor formation.
Previous study identified the dysregulation of miR-27b-3p in many diseases. For example, miR-27b-3p expression was decreased in oral lichen planus samples [23] and chronic liver injury [24]. miR-27b-3p was dramatically decreased in gastric cancer tissue as well as cell samples, and its introduction inhibited cell growth [25]. Herein, mechanism assay confirmed miR-27b-3p bound to circRANBP17. Following that, our data indicated the direct binding relationship of miR-27b-3p with circRANBP17. A comparative qRT-PCR analysis about NB tissues and cells confirmed miR-27b-3p was apparently decreased in clinical NB specimens and cell samples. miR-27b-3p expression was associated with tumor-node-metastasis grade. Meanwhile, it was found that circRANBP17 expression was negatively correlated with miR-27b-3p in clinical NB specimens. Moreover, the further functional study manifested that miR-27b-3p inhibitor could reverse circRANBP17-induced inhibition of cell proliferation and metastasis, and promotion of apoptosis.
KDM1A also reported to promote tumorigenesis in many cancers. For example, Pishas et al. explained KDM1A was obviously increased in Ewing sarcoma specimens and cells in comparison with controls [26]. In a study about prostate cancer, we found KDM1A was conspicuously increased in relapse prostate cancer tissues, and KDM1A silencing inhibited cell proliferation [27]. Actually, Yang et al. demonstrated KDM1A expression was distinctly enhanced in NB tissues, and KDM1A overexpression reversed the increased cell proliferation and metastasis caused by miR-329 [17]. Additionally, KDM1A combined with miR-542-3p to promote NB cell proliferation and invasion [28]. Here, we confirmed KDM1A was strikingly augmented in clinical NB specimens and cell samples. Mechanism assay validated KDM1A bound to miR-27b-3p. Meanwhile, KDM1A was negatively linear correlated with miR-27b-3p. Furthermore, the functional exploration indicated that KDM1A overexpression mitigated the decreased cell proliferative and metastatic capacities and increased apoptotic rate by miR-27b-3p in NB cells. In addition, in vivo data showed circRANBP17 knockdown repressed KDM1A production. Importantly, the mechanistic analysis revealed that cir-cRANBP17 regulated KDM1A expression via absorbing miR-27b-3p in NB cells.

Conclusion
We found that circRANBP17 and KDM1A were remarkably up-increased, while miR-27b-3p was drastically Figure 7: KDM1A overexpression mitigated the inhibitory effects on cell proliferation, migration, invasion and the promotion effect on cell apoptosis in SK-N-SH and IMR-32 cells by targeting miR-27b-3p. Cell proliferative capacity (a-d), migration ability (e and f), invasion ability (g), apoptosis rate (h) and the protein levels of Bcl-2, Bax and Cleaved caspase 3 (i and j) in SK-N-SH and IMR-32 cells transfected with miR-27b-3p, miR-27b-3p + KDM1A or their matched controls were assessed by MTT assay, EdU assay, cell colony formation assay, Transwell assay, flow cytometry and western blot, respectively. *P < 0.05. decreased in clinical NB specimens and cell samples. Based on the all results, we found that circRANBP17 positively modulated KDM1A through binding to miR-27b-3p, thus regulating tumor progression in NB. Conflict of interest: The authors declare that they have no financial conflict of interest.
Data availability statement: The original western blots pictures and datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. (c-e) Impacts of circRANBP17 silencing on the expression of circRANBP17, miR-27b-3p and KDM1A were determined by qRT-PCR or western blot in the forming tumors from sh-circRANBP17 or sh-NC group. (f) Positive expression rates of KDM1A and Ki-67 were evaluated by IHC assay. *P < 0.05.